The present invention relates to a method and kit for detecting, or determining the quantity of, β-lactam penicillins, as well as, haptens, immunogens, conjugates and antibodies useful therein.
By “detecting” is meant qualitatively analysing for the presence or absence of a substance.
By “determining” is meant quantitatively analysing for the amount of a substance.
The present invention is intended to have broad applicability across the main first generation β-lactam penicillins such as ampicillin, penicillin G, amoxycillin, cloxacillin, dicloxacillin and oxacillin, but is not intended to be limited to these specific β-lactam penicillins.
Antibiotics are routinely used in animal husbandry for both prophylactic and therapeutic purposes. The β-lactam class of antibiotics are commonly used in the meat and dairy industry, as growth enhancers. This class, also known as the penicillins, is used to treat dairy cow mastitis, thereby increasing milk yields and the productive life span of the cow. β-lactams may also be included in animal feed, with the purpose of enhancing the growth of poultry and pigs. Through disease prevention, or by inhibiting the activity of natural gut flora in such animals, the antibiotic causes the animals to reach marketable size faster than without the use of such enhancers.
Problems, however, may arise when residues of the β-lactams are present in meat and dairy products. As with any antibiotic, continuous exposure of humans to the β-lactams can result in a reduction of the efficiency of the drugs used to treat diseases, due to the development of resistant strains of pathogenic bacteria. The presence of the β-lactams in consumed food may also result in allergic reactions in penicillin sensitive humans. Dairy products containing these antibiotics may also interfere with bacterial cultures used in processing.
As a result, strict guidelines have been imposed throughout the European Community regarding withdrawal times and the maximum recommended levels (MRLs) of the β-lactams in milk and meat. Milk and meat samples are routinely tested to ensure that they comply with this EC legislation. Various methods are used to test for antibiotics such as the β-lactams. Many of these tests are based on microbial inhibition tests, which are time consuming and may be specific for individual β-lactams. The development of a method for the rapid detection of the β-lactams in milk and meat would be especially valuable if the method was generic, i.e., detected most, if not all, β-lactam antibiotics.
Many attempts have been made to raise antibodies in β-lactam sensitised animals, with the aim of producing an immunoassay for the detection of β-lactams in general. The first stage of such a process is to produce an immunogen which will elicit an immune response in the animal host. This is problematic due to the failure of the β-lactam ring to remain intact during conjugation to a carrier protein. Known conjugation methods based on the open lactam ring are disclosed in, for example, U.S. Pat. No. 4,347,312, U.S. Pat. No. 5,128,240, in de Haan et al, 1985 and in Faghihi Shirazi et al, 1991. This results in the production of antisera sensitive to the open form of the β-lactam ring, which may not necessarily be sensitive to the generic ring structure.
Alternatively, for example, the free carboxyl group of the closed β-lactam ring may be esterified, as is disclosed in EP-A-309,299 and in Usleber et al, 1994. Antisera raised to β-lactam antibiotics conjugated in this manner are specific for the acyl side chains and only cross react with other β-lactam antibiotics if they have similar side chains, as is the case with the isoxazolyl penicillins. Further, alternatively, conjugation can occur by way of the 6-amino group of 6-amino penicillanic acid, as is disclosed in EP-A-309,299 and de Leuw et al, 1997. In such cases, where the β-lactam ring remains intact during conjugation, the antibodies display high cross reactivity with the main first generation β-lactams. A further potential conjugation site for β-lactam penicillins is via the a amino group of the D-α-aminoacetamido group of penicillin, as is disclosed in, for example, Nagakura et al, 1991. The cell lines disclosed in Nagakura et al, 1991, Abp4 and Abp7, concern haptens and conjugates using a MBS (maleimidobenzoyl-N-hydroxysuccinimide) cross-linker and the document concludes that one of the cell lines (Abp4) recognises the thiazolidine ring whilst the other of the cell lines (Abp7) recognises the acyl side chain. Abp4 cross reacts with penicillin G, 6-aminopenicillanic acid and certain cephalosporins, while Abp7 is highly specific, displaying little or no cross reactivity with the main first generation β-lactams.